High affinity humanized anti-tag-72 monoclonalantibodies

ABSTRACT

Novel humanized monoclonal antibodies, humanized antibody fragments, and derivatives thereof which specifically bind TAG-72 are provided as well as methods for their manufacture. These humanized antibodies are useful in the treatment of cancers which express TAG-72 as well as for diagnostic purposes, e.g., for in vivo imaging of tumors or cancer cells which express TAG-72.

FIELD OF THE INVENTION

[0001] The present invention relates to humanized monoclonal antibodiesand fragments or derivatives thereof which specifically bindtumor-associated glycoprotein TAG-72, a human pancarcinoma antigenexpressed by various human tumor cells. More specifically, the presentinvention relates to humanized monoclonal antibodies and fragments orderivatives thereof derived from murine monoclonal antibody CC49 orother murine antibodies which specifically bind TAG-72. The presentinvention further relates to methods for producing such humanizedmonoclonal antibodies specific to TAG-72, pharmaceutical and diagnosticcompositions containing such humanized monoclonal antibodies, andmethods of use thereof for the treatment or diagnosis of cancer.

BACKGROUND OF THE INVENTION

[0002] The identification of antigens expressed by tumor cells and thepreparation of monoclonal antibodies which specifically bind suchantigens is well known in the art. Anti-tumor monoclonal antibodiesexhibit potential application as both therapeutic and diagnostic agents.Such monoclonal antibodies have potential application as diagnosticagents because they specifically bind tumor antigens and thereby candetect the presence of tumor cells or tumor antigen in an analyte. Forexample, use of monoclonal antibodies which bind tumor antigens for invitro and in vivo imaging of tumor cells or tumors using a labeled formof such a monoclonal antibody is conventional in the art.

[0003] Moreover, monoclonal antibodies which bind tumor antigens havewell known application as therapeutic agents. The usage of monoclonalantibodies themselves as therapeutic agents, or as conjugates whereinthe monoclonal antibody is directly or indirectly attached to aneffector moiety, e.g., a drug, cytokine, cytotoxin, etc., is well known.

[0004] Essentially, if the monoclonal antibody is attached to aneffector moiety the monoclonal antibody functions as a targeting moiety,i.e. it directs the desired effector moiety (which typically possessestherapeutic activity) against a desired target, e.g., a tumor whichexpresses the antigen bound by the monoclonal antibody. In contrast,when the monoclonal antibody itself operates as a therapeutic agent, theantibody functions both as a targeting moiety—i.e., it will specificallybind a cell which expresses the antigen—and as an effector whichmediates therapeutic activity, typically tumor cell lysis. Such effectorfunctions—including, e.g., antibody-dependent cellular cytotoxicity(ADCC) and complement dependent cytotoxicity (CDC), among others—areeffected by the portion of the antibody molecule generally referred toin the literature as the Fc portion. One specific tumor antigen againstwhich various monoclonal antibodies have been developed istumor-associated glycoprotein TAG-72. TAG-72 is expressed on the surfaceof various human tumor cells, such as the LS174T tumor cell line(American Type Tissue Collection (ATCC) No. CL188, a variant of cellline LS180 (ATCC No. CL187)), a colon adenocarcinoma line. Variousresearch groups have reported the production of monoclonal antibodies toTAG-72. See, e.g., Muraro et al., Cancer Res., 48:4588-4596 (1988);Johnson et al., Cancer Res., 46:850-857 (1986); Molinolo et al., CancerRes., 50:1291-1298 (1990); Thor et al., Cancer Res., 46:3118-3127(1986); EP 394,277 to Schlom et al. (assigned to the National CancerInstitute); and U.S. Pat. No. 5,512,443 to Jeffrey Schlom et al.Specific antibodies to TAG-72 which are publicly available include CC49(ATCC No. HB 9459), CC83 (ATCC No. HB 9453), CC46 (ATCC No. HB 9458),CC92 (ATCC No. HB 9454), CC30 (ATCC No. HB 9457), CC11 (ATCC No. 9455),and CC15 (ATCC No. HB 9460).

[0005] One example thereof, CC49, is a murine monoclonal antibody of theIgG_(I) isotype. This monoclonal antibody is a second generationmonoclonal antibody prepared by immunizing mice with TAG-72 purifiedusing the first generation antibody B72.3. Colcher et al., Proc. Natl.Acad. Sci. USA, 78:3199-3203 (1981). CC49 specifically binds TAG-72, andhas a higher antigen-binding affinity than B72.3. Muraro et al., CancerRes., 48:4588-4596 (1988). This monoclonal antibody has been reported totarget human colon carcinoma xenografts efficiently, and to reduce thegrowth of such xenografts with good efficacy. Molinolo et al, CancerRes., 50:1291-1298 (1996); Colcher et al., J. Natl. Cancer Inst.,82:1191-1197 (1990). Also, radiolabeled CC49 has been reported toexhibit excellent tumor localization in several ongoing clinical trials.

[0006] However, while murine antibodies have applicability astherapeutic agents in humans, they are disadvantageous in some respects.Specifically, murine antibodies, because of the fact that they are offoreign species origin, may be immunogenic in humans. This may result ina neutralizing antibody response (human anti-murine antibody (HAMA)response), which is particularly problematic if the antibodies aredesired to be administered repeatedly, e.g., in treatment of a chronicor recurrent disease condition. Also, because they contain murineconstant domains they may not exhibit human effector functions.

[0007] In an effort to eliminate or reduce such problems, chimericantibodies have been disclosed. Chimeric antibodies contain portions oftwo different antibodies, typically of two different species. Generally,such antibodies contain human constant regions and variable regions ofanother species, typically murine variable regions. For example, somemouse/human chimeric antibodies have been reported which exhibit bindingcharacteristics of the parental mouse antibody, and effector functionsassociated with the human constant region. See, e.g.: U.S. Pat. No.4,816,567 to Cabilly et al.; U.S. Pat. No. 4,978,745 to Shoemaker etal.; U.S. Pat. No. 4,975,369 to Beavers et al.; and U.S. Pat. No.4,816,397 to Boss et al. Generally, these chimeric antibodies areconstructed by preparing a genomic gene library from DNA extracted frompre-existing murine hybridomas. Nishimura et al., Cancer Res., 47:999(1987). The library is then screened for variable region genes from bothheavy and light chains exhibiting the correct antibody fragmentrearrangement patterns. Alternatively, cDNA libraries are prepared fromRNA extracted from the hybridomas and screened, or the variable regionsare obtained by polymerase chain reaction. The cloned variable regiongenes are then ligated into an expression vector containing clonedcassettes of the appropriate heavy or light chain human constant regiongene. The chimeric genes are then expressed in a cell line of choice,usually a murine myeloma line. Such chimeric antibodies have been usedin human therapy.

[0008] Moreover, the production of chimeric mouse-human antibodiesderived from CC49 and CC83, which specifically bind TAG-72, has beenreported. In this regard, see e.g., EPO 0,365,997 to Mezes et al. (TheDow Chemical Company). One such chimeric CC49 antibody is that producedby the cell line deposited as ATTC No. HB 9884 (Budapest).

[0009] Also, Morrison et al. report the preparation of several antitumorchimeric monoclonal antibodies, in Important Advances in Oncology,Recombinant Chimeric Monoclonal Antibodies, pp. 3-18 (S. A. Rosenberg,ed., 1990) (J. B. Lippincott, Philadelphia, Pa.). Results of clinicaltrials with chimeric cMAb-17-1A in patients with metastatic colorectalcarcinoma now show that this antibody has a 6-fold longer circulationtime and significantly reduced immunogenicity as compared to the murinemonoclonal antibody from which it was derived. LoBuglio et al., Proc.Natl. Acad. Sci. USA, 86:4220-4224 (1989); Meredith et al., J. Nucl.Med., 32:1162-1168 (1991).

[0010] However, while such chimeric monoclonal antibodies typicallyexhibit lesser immunogenicity, they are still potentially immunogenic inhumans because they contain murine variable sequences which may elicitantibody responses. Thus, there is the possibility that these chimericantibodies may elicit an anti-idiotypic response if administered topatients. Saleh et al., Cancer Immunol. Immunother., 32:185-190 (1990).

[0011] For example, when cB72.3(γ4) was administered to patients withcolorectal carcinomas, 62% of such patients elicited a human antimurineantibody (HAMA) response, which included an anti-V-region response. Thisis disadvantageous because a HAMA response would make repeated antitumorantibody administration potentially ineffective because of an increasedantibody clearance from the serum (Saleh et al., Cancer Immunol.Immunother., 32:180-190 (1990)) and also because of potential allergicreactions (LoBuglio et al., Hybridoma, 5:5117-5123 (1986).

[0012] A number of genetic variants of potential clinical utility havebeen developed from MAb CC49. These include cCC49, a C_(H)2domain-deficient CCC49 (Slavin-Chiorini et al, Int. J Cancer, 53:97-103(1993)), and a single chain Fv (sFv) (Milenic et al., Cancer Res.,51:6365-6371 (1991); Sawyer et al., Protein Eng., 7:1401-1406 (1994)).These molecules may elicit relatively reduced HAMA responses inpatients, since they have shown more rapid plasma and whole bodyclearance rates in mice and rhesus monkeys, as compared to intact IgG.Slavin-Chiorini et al. (1993) (id.); Milenic et al. (1991) (id.).Additionally, novel single-chain immunoglobulin (SClg) molecules derivedfrom cCC49 have been reported and are encoded by single-gene constructs.One such molecule, SCIgΔC_(H)1 consists of CC49 sFv linked to the humanγ1 Fc region (Shu et al., Proc. Natl. Acad. Sci. USA, 90:7915-7999(1993)) while the other SCIg-IL-2 carries a human interleukin-2 (IL-2)molecule genetically attached to the carboxyl end of the Fc region ofSCIgΔC_(H)1 (Kashmiri et al., Proc. XVI Intl. Cancer Cong., 1:183-187(1994)). Both SCIgs are comparable to cCC49 in antigen binding andantibody cellular cytolytic activity. The biological activity of theIL-2 is also retained in SCIg-IL-2.

[0013] In an effort to alleviate the immunogenicity concerns of chimericand murine antibodies, the production of “humanized” antibodies is alsoknown. Ideally, “humanization” results in an antibody that isnon-immunogenic in humans, with substantially complete retention of theantigen-binding properties of the original molecule. In order to retainall the antigen-binding properties of the original antibody, thestructure of its combining-site has to be faithfully reproduced in the“humanized” version. This can potentially be achieved by transplantingthe combining site of the nonhuman antibody onto a human framework,either (a) by grafting only the nonhuman CDRs onto human framework andconstant regions with or without retention of critical frameworkresidues (Jones et al., Nature, 321:522 (1986); Verhoeyen et al.,Science, 239:1539 (1988)); or (b) by transplanting the entire nonhumanvariable domains (to preserve ligand-binding properties) but also“cloaking” them with a human-like surface through judicious replacementof exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol.,28:489 (1991)).

[0014] Essentially, humanization by CDR grafting involves transplantingonly the CDRs onto human fragment and constant regions. Theoretically,this should substantially eliminate immunogenicity (except if allotypicor idiotypic differences exist). Jones et al., Nature, 321:522-525(1986); Verhoeyen et al., Science, 239:1534-1536 (1988); Riechmann etal., Nature, 332:323-327 (1988). While such a technique is effective insome instances, CDR-grafting sometimes does not yield the desiredresult. Rather, it has been reported that some framework residues of theoriginal antibody may also need to be preserved in order to preserveantigen binding activity. Riechmann et al., Nature, 332:323-327 (1988);Queen et al., Proc. Natl. Acad. Sci. USA, 86:10023-10029; Tempest etal., Biol. Technology, 9:266-271 (1991); Co et al., Nature, 351:501 -502(1991)).

[0015] As discussed, in order to preserve the antigen-binding propertiesof the original antibody, the structure of its combining site must befaithfully reproduced in the humanized molecule. X-ray crystallographicstudies have shown that the antibody combining site is built primarilyfrom CDR residues, although some neighboring framework residues havebeen found to be involved in antigen binding. Amit et al., Science,233:747-753 986); Colman et al., Nature, 326:358-363 (1987); Sheriff etal., Proc. Natl. Acad. Sci. USA, 84:8075-8079 (1987); Padlan et al.,Proc. Natl. Acad Sci. USA, 86:5938-5942 (1989); Fischmann et al., J.Biol. Chem., 266:12915-12920 (1991); Tulip et al., J. Molec. Biol.,227:122-148 (1992). It has also been found that the structures of theCDR loops are significantly influenced by surrounding frameworkstructures. Chothia et al., J. Molec. Biol., 196:901-917 (1987); Chothiaet al., Nature, 342:877-883 (1989); Tramomonteno et al., J. Molec. BioL,215:175-182 (1990).

[0016] Small but significant differences in the relative disposition ofthe variable light chain (V_(L)) and variable heavy (V_(H)) domains havebeen noted (Colman et al., Nature, 326:358-363 (1987)) and thosedifferences are ostensibly due to variations in the residues involved inthe interdomain contact (Padlan et al., Molec. Immunol., 31:169-217(1994)).

[0017] Furthermore, structural studies of the effect of the mutation ofinterior residues, in which changes in side chain volume are involved,have shown that the resulting local deformations are accommodated byshifts in side chain positions that are propagated to distant parts ofthe molecular interior. This suggests that during humanization theinterior residues in the variable domains and in the interface betweenthese domains, or at least the interior volumes, should also bemaintained; a humanization protocol in which an interior residue isreplaced by one of different physical properties (such as size, charge,or hydrophobicity, etc.), could result in a significant modification ofthe antigen combining site structure.

[0018] One method of identifying the framework residues which need to bepreserved is by computer modeling. Alternatively, critical frameworkresidues may potentially be identified by comparing known antibodycombining site structures (Padlan, Molec. Immun., 31(3):169-217 (1994)).

[0019] The residues which potentially affect antigen binding fall intoseveral groups. The first group comprises residues that are contiguouswith the combining site surface and which could therefore make directcontact with antigens. They include the amino-terminal residues andthose adjacent to the CDRs. The second group includes residues thatcould alter the structure or relative alignment of the CDRs either bycontacting the CDRs or the opposite chains. The third group comprisesamino acids with buried side chains that could influence the structuralintegrity of the variable domains. The residues in these groups areusually found in the same positions (ibid.) according to the adoptednumbering system. See Kabat et al., Sequences of Proteins ofImmunological Interest, NIH Pub. No. 91-3242 (5th ed., 1991) (U.S. Dept.Health & Human Services, Bethesda, Md.) and Genbank.

[0020] However, while humanized antibodies are desirable because oftheir potential low immunogenicity in humans, their production isunpredictable. For example, sequence modification of antibodies mayresult in substantial or even total loss of antigen binding affinity, orloss of binding specificity. Alternatively, “humanized antibodies” maystill exhibit immunogenicity in humans, irrespective of sequencemodification.

[0021] Thus, there still exists a significant need in the art for novelhumanized antibodies to desired antigens. More specifically, thereexists a need in the art for humanized antibodies specific to TAG-72,because of their potential as immunotherapeutic and immunodiagnosticagents.

OBJECTS OF THE INVENTION

[0022] Toward this end, it is an object of the invention to providehumanized antibodies which are specific to human TAG-72.

[0023] More specifically, it is an object of the invention to providehumanized antibodies derived from murine antibodies to TAG-72, and inparticular from CC49, a specific murine antibody which binds to TAG-72.

[0024] It is also an object of the invention to provide pharmaceuticalcompositions containing humanized antibodies which are specific toTAG-72. It is a more specific object of the invention to providepharmaceutical compositions containing humanized antibodies derived fromCC49, a murine antibody which specifically binds to TAG-72.

[0025] It is another specific object of the invention to provide methodsof using humanized antibodies to TAG-72 for treatment of cancers whichexpress TAG-72, in particular human colon cancer.

[0026] It is another object of the invention to provide immunodiagnosticcompositions for detecting cancer cells which contain a humanizedantibody which specifically binds TAG-72, and preferably is derived fromCC49, which antibody is in labeled or unlabeled form. It is anotherobject of the invention to provide a method of immunodiagnosis of cancerusing compositions which contain a humanized antibody which specificallybinds TAG-72, which is in labeled or unlabeled form.

[0027] It is still another object of the invention to provide nucleicacid sequences which encode for humanized antibodies to TAG-72 orfragments thereof. It is a more specific object of the invention toprovide nucleic acid sequences which encode humanized antibodies derivedfrom CC49, a murine antibody which specifically binds to TAG-72. It isanother object of the invention to provide vectors from which may beexpressed humanized antibodies to TAG-72, in particular humanizedantibodies derived from CC49, a murine antibody which specifically bindsto TAG-72.

BRIEF DESCRIPTION OF THE FIGS.

[0028]FIG. 1 aligns amino acid sequences of murine CC49 V_(H), the NEWMframework regions encoded by the FR starting material, and the humanizedNEWM-based V_(H) (HuVH) disclosed in Example 1. The CDRs are in theboxes. Murine residues retained in the FRs are identified with arrowsymbols (↑). Murine FR residues retained in alternate versions of theHuVH are identified with letter symbols (A), (S), and (K).

[0029]FIG. 2 aligns amino acid sequences of murine CC49 V_(K), the REIframework regions encoded by the FR starting material, and the humanizedREI-based V_(K) disclosed in Example 1. CDRs are in the boxes.

[0030]FIG. 3 aligns the variable heavy chain of CC49, the HuCC49disclosed in Example 1, and NEWM.

[0031]FIG. 4 aligns the variable light chain of CC49, the HuCC49disclosed in Example 1, and REI.

[0032]FIG. 5 contains schematics of the vectors used to express thehumanized V_(H) and V_(K) shown in FIG. 3 and FIG. 4.

[0033]FIG. 6 is an ELISA showing binding of CC49 antibodies HuVHA/MuVKand HuVHA/HuVK to TAG-72.

[0034]FIG. 7 is an ELISA showing binding of CC49 antibodies MuVH/MuVKand HuVH/HuVK to TAG-72.

[0035]FIG. 8 is an ELISA showing binding of CC49 antibodies MuVH/MuVKand HuVH/HuVK to TAG-72.

[0036]FIG. 9 is an ELISA showing binding of CC49 antibodies MuVH/MuVKand HuVHA/HuVK to TAG-72.

[0037]FIG. 10 is an ELISA showing binding of CC49 antibodies HuVH/HuVKand HuVHK/HuVK to TAG-72.

[0038]FIG. 11 is an ELISA showing binding of CC49 antibodies HuVHS/HuVKand HuVH/HuVK to TAG-72.

[0039]FIG. 12 is a Scatchard analysis of humanized (HuVH/HuVK) andchimeric (MuVH/MuVK) CC49 monoclonal antibodies.

[0040]FIG. 13 presents the single-stranded DNA sequence of the templateused to produce the initial humanized NEWM-based VHs, HuVH and HuVHA.

[0041]FIG. 14 presents the double-stranded DNA sequence of the templateused to produce the alternate humanized VHs, HuVHS and HuVHK.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Prior to setting forth the invention, definitions of certainterms which are used in this disclosure are set forth below:

[0043] Antibody—This refers to single chain, two-chain, and multi-chainproteins and glycoproteins belonging to the classes of polyclonal,monoclonal, chimeric, and hetero immunoglobulins (monoclonal antibodiesbeing preferred); it also includes synthetic and genetically engineeredvariants of these immunoglobulins. “Antibody fragment” includes Fab,Fab', F(ab')₂, and Fv fragments, as well as any portion of an antibodyhaving specificity toward a desired target epitope or epitopes.

[0044] Humanized antibody—This will refer to an antibody derived from anon-human antibody, typically murine, that retains or substantiallyretains the antigen-binding properties of the parent antibody but whichis less immunogenic in humans. This may be achieved by various methodsincluding (a) grafting only the non-human CDRs onto human framework andconstant regions with or without retention of critical frameworkresidues, or (b) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues. Such methods as are useful in practicing the present inventioninclude those disclosed in Jones et al., Morrison et al., Proc. NatLAcad. Sci. USA, 81:6851 -6855 (1984); Morrison and Oi, Adv. Immunol.,44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988);Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,31(3):169-217 (1994).

[0045] Complementarity Determining Region. or CDR—The term CDR, as usedherein, refers to amino acid sequences which together define the bindingaffinity and specificity of the natural Fv region of a nativeimmunoglobulin binding site as delineated by Kabat et al. (1991).

[0046] Framework Region—The term FR, as used herein, refers to aminoacid sequences interposed between CDRs. These portions of the antibodyserve to hold the CDRs in an appropriate orientation for antigenbinding. In the antibodies and antibody fragments of the presentinvention, the framework regions for the light chain variable region maybe selected from the group consisting of human lambda light chain FRsand human kappa subgroup I, II, and III light chain FRs, whethercomprising their fully human native amino acid sequences or comprisingamino acid sequence modifications necessary to retain or increasebinding affinity and/or binding specificity.

[0047] Constant Region—The portion of the antibody molecule whichconfers effector functions. In the present invention, murine constantregions are substituted with human constant regions. The constantregions of the subject chimeric or humanized antibodies are derived fromhuman immunoglobulins. The heavy chain constant region can be selectedfrom any of the five isotypes: alpha, delta, epsilon, gamma or mu.Further, heavy chains of various subclasses (such as the IgG subclassesof heavy chains) are responsible for different effector functions andthus, by choosing the desired heavy chain constant region chimericantibodies with desired effector function can be produced.

[0048] Preferred constant regions are gamma 1 (IgG1), gamma 3 (IgG3) andgamma 4 (IgG4). More preferred is a constant region of the gamma 1(IgG1) isotype. The light chain constant region can be of the kappa orlambda type, preferably of the kappa type.

[0049] Chimeric antibody—This is an antibody containing sequencesderived from two different antibodies, which typically are of differentspecies. Most typically chimeric antibodies comprise human and murineantibody fragments, generally human constant and murine variableregions.

[0050] Mammals—Animals that nourish their young with milk secreted bymammary glands, preferably warm blooded mammals, more preferably humans.

[0051] Immunogenicity—A measure of the ability of a targeting protein ortherapeutic moiety to elicit an immune response (humoral or cellular)when administered to a recipient. The present invention is concernedwith the immunogenicity of the subject humanized antibodies or fragmentsthereof.

[0052] Humanized antibody of reduced immunogenicity—This refers to ahumanized antibody exhibiting reduced immunogenicity relative to theparent antibody.

[0053] Humanized antibody substantially retaining the binding propertiesof the parent antibody—This refers to a humanized antibody which retainsthe ability to specifically bind the antigen recognized by the parentantibody used to produce such humanized antibodies. Preferably thehumanized antibody will exhibit the same or substantially the sameantigen-binding affinity and avidity as the parent antibody, e.g., CC49.Preferably, the affinity of the antibody will at least about 10% of thatof the parent antibody. More preferably, the affinity will be at leastabout 25%, i.e. at least two-fold less than the affinity of the parentantibody. Most preferably the affinity will be at least about 50% thatof the parent antibody. Methods for assaying antigen-binding affinityare well known in the art and include half-maximal binding assays,competition assays, and Scatchard analysis. Suitable antigen bindingassays are described in this application.

[0054] In its broadest embodiment, the present invention is directed tohumanized antibodies which specifically bind TAG-72, a pancarcinomaantigen expressed by various human cancers, in particular human coloncarcinoma. Preferably, such humanized antibodies will be derived fromantibodies having good binding affinity to TAG-72, e.g.: B72.3 (Thor etal., Cancer Res., 46:31 18-3127 (1986); Johnson et al., Cancer Res.,46:850-857 (1986)), deposited as ATCC No. HB 8108; CC49 (ATCC No. HB9459); CC83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9452); CC92 (ATCC No.HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No. 9455); and CC15 (ATCCNo. HB 9460); or chimerized forms thereof (see, e.g., EPO 0,365,997 toMezes et al., The Dow Chemical Company).

[0055] Most preferably, such humanized antibodies will be derived fromCC49, which has been reported to target human colon carcinoma xenograftsefficiently and also to reduce the growth of the xenograft with goodefficacy. Molinolo et al., Cancer Res., 50:1291 -1298 (1990); Colcher etal., J. Natl. Cancer Inst., 82:1191 -1197 (1990).

[0056] As discussed above, humanized antibodies afford potentialadvantages over murine and also chimeric antibodies, e.g., reducedimmunogenicity in humans. This is advantageous because it should reduceand potentially eliminate the eliciting of a HAMA response when suchhumanized antibodies are administered in vivo, e.g., for treatment ofcancer or for diagnosis of cancer, e.g., for tumor imaging.

[0057] However, as noted, humanization may in some instances adverselyaffect antigen binding. Preferably, the humanized antibodies of thepresent invention which specifically bind TAG-72 will possess a bindingaffinity for TAG-72 of at least about 10% and more preferably at leastabout 25% and most preferably at least about 50% that of the TAG-72antigen binding affinity of the parent murine antibody, e.g., B72.3,CC49, CC46, CC30,CC11, CC15, CC83, or another parent antibody. Mostpreferably, the humanized antibodies of the present invention willpossess a binding affinity for TAG-72 of at least about 10% and morepreferably at least about 25% and most preferably at least about 50%that of the TAG-72 antigen binding affinity of either CC49 or a chimericCC49 antibody.

[0058] Preferably, the humanized antibodies of the present inventionwill bind the same epitope as CC49. Such antibodies can be identifiedbased on their ability to compete with CC49 for binding to TAG-72 or toTAG-72-expressing cancer cells.

[0059] In general, the subject humanized antibodies are produced byobtaining nucleic acid sequences encoding the variable heavy andvariable light sequences of an antibody which binds TAG-72, preferablyCC49, identifying the CDRs in said variable heavy and variable lightsequences, and grafting such CDR nucleic acid sequences onto humanframework nucleic acid sequences.

[0060] Preferably, the selected human framework will be one that isexpected to be suitable for in vivo administration, i.e., does notexhibit immunogenicity. This can be determined, e.g., by priorexperience with in vivo usage of such antibodies and by studies of aminoacid sequence similarities. In the latter approach, the amino acidsequences of the framework regions of the antibody to be humanized,e.g., CC49, will be compared to those of known human framework regions,and human framework regions used for CDR grafting will be selected whichcomprise a size and sequence most similar to that of the parentantibody, e.g., a murine antibody which binds TAG-72. Numerous humanframework regions have been isolated and their sequences reported in theliterature. See, e.g., Kabat et al., (id.).This enhances the likelihoodthat the resultant CDR-grafted “humanized” antibody, which contains theCDRs of the parent (e.g., murine) antibody grafted onto the selectedhuman framework regions will significantly retain the antigen bindingstructure and thus the binding affinity of the parent antibody. As aresult of such studies, the FRs of REI and NEWM antibodies have beenidentified as having amino acid sequences which are likely to allow theCDRs of CC49 to retain a significant degree of antigen binding affinity.As noted, the selected human framework regions will preferably be thosethat are expected to be suitable for in vivo administration, i.e., notimmunogenic. Based on their amino acid sequences, REI and NEWM humanframework regions are expected to be substantially non-immunogenic.

[0061] Methods for cloning nucleic acid sequences encodingimmunoglobulins are well known in the art. Such methods will generallyinvolve the amplification of the immunoglobulin sequences to be clonedusing appropriate primers by polymerase chain reaction (PCR). Primerssuitable for amplifying immunoglobulin nucleic acid sequences, andspecifically murine variable heavy and variable light sequences havebeen reported in the literature. After such immunoglobulin sequenceshave been cloned, they will be sequenced by methods well known in theart. This will be effected in order to identify the variable heavy andvariable light sequences, and more specifically the portions thereofwhich constitute the CDRs and FRs. This can be effected by well knownmethods.

[0062] Once the CDRs and FRs of the cloned antibody sequences which areto be humanized have been identified, the amino acid sequences encodingCDRs are then identified (deduced based on the nucleic acid sequencesand the genetic code and by comparison to previous antibody sequences)and the corresponding nucleic acid sequences are grafted onto selectedhuman FRs. This may be accomplished by use of appropriate primers andlinkers. Methods for selecting suitable primers and linkers to providefor ligation of desired nucleic acid sequences is well within thepurview of the ordinary artisan and include those disclosed in U.S. Pat.No. 4,816,397 to Boss et al. and U.S. Pat. No. 5,225,539 to Winter etal.

[0063] After the CDRs are grafted onto selected human FRs, the resultant“humanized” variable heavy and variable light sequences will then beexpressed to produce a humanized Fv or humanized antibody which bindsTAG-72. Typically, the humanized variable heavy and variable lightsequences will be expressed as a fusion protein with human constantdomain sequences, so that an intact antibody which binds TAG-72 isobtained. However, this is not necessary as the variable heavy and lightsequences can also be expressed in the absence of constant sequences toproduce a humanized Fv which binds TAG-72. However, fusion of humanconstant sequences to the humanized variable region(s) is potentiallydesirable because the resultant humanized antibody which binds TAG-72will then possess human effector functions such as complement-dependentcytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)activity. Such activity has been found in chimeric antibodies, includingCC49. The humanized anti-TAG-72 antibodies of the present invention canalso support such effector function activity with the added advantage ofa greatly decreased risk of a HAMA response.

[0064] Methods for synthesizing DNAs encoding a protein of knownsequence are well known in the art. Using such methods, DNA sequenceswhich encode the subject humanized V_(L) and V_(H) sequences aresynthesized, and then expressed in vector systems suitable forexpression of recombinant antibodies. This may be effected in any vectorsystem which provides for the subject humanized V_(L) and V_(H)sequences to be expressed as a fusion protein with human constant domainsequences and to associate to produce functional (antigen binding)antibodies. Expression vectors and host cells suitable for expression ofrecombinant antibodies and humanized antibodies in particular, are wellknown in the art.

[0065] The following references are representative of methods andvectors suitable for expression of recombinant immunoglobulins which maybe utilized in carrying out the present invention. Weidle et al., Gene,51:21 -29 (1987); Dorai et al., J. Immunol., 13(12):4232-4241 (1987); DeWaele et al., Eur. J. Biochem., 176:287-295 (1988); Colcher et al.,Cancer Res., 49:1738-1745 (1989); Wood et al., J. Immunol, 145(a):3011-3016 (1990); Bulens et al., Eur. J. Biochem., 195:235-242 (1991);Beggington et al., Biol. Technology, 10:169 (1992); King et al.,Biochem. J., 281:317-323 (1992); Page et al., Biol. Technology, 9:64(1991); King et al., Biochem. J., 290:723-729 (1993); Chaudary et al.,Nature, 339:394-397 (1989); Jones et al., Nature, 321:522-525 (1986);Morrison and Oi, Adv. ImmunoL, 44:65-92 (1988); Benhar et al., Proc.Natl. Acad. Sci. USA, 91:12051-12055 (1994); Singer et al., J. Immunol.,150:2844-2857 (1993); Cooto et al., Hybridoma, 13(3):215-219 (1994);Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989); Caronet al., Cancer Res., 32:6761 -6767 (1992); Cotoma et al., J. Immunol.Meth., 152:89-109 (1992). Moreover, vectors suitable for expression ofrecombinant antibodies are commercially available. The vector may, e.g.,be a bare nucleic acid segment, a carrier-associated nucleic acidsegment, a nucleoprotein, a plasmid, a virus, a viroid, or atransposable element.

[0066] Host cells known to be capable of expressing functionalimmunoglobulins include, e.g.: mammalian cells such as Chinese HamsterOvary (CHO) cells; COS cells; myeloma cells, such as NSO and SP2/0cells; bacteria such as Escherichia coli; yeast cells such asSaccharomyces cerevisiae; and other host cells. Of these, CHO cells areused by many researchers given their ability to effectively express andsecrete immunoglobulins. NSO cells are one of the preferred types ofhost cells useful in the present invention.

[0067] Essentially, recombinant expression of humanized antibodies iseffected by one of two general methods. In the first method, the hostcells are transfected with a single vector which provides for theexpression of both heavy and light variable sequences optionally fusedto selected constant regions. In the second method, host cells aretransfected with two vectors, each of which encodes a different variablechain (i.e. a variable heavy chain or variable light chain); eachvariable chain-encoding vector may optionally provide for the expressionof the variable chain fused to a selected constant region.

[0068] Human constant domain sequences are well known in the art, andhave been reported in the literature. Preferred human light chainconstant sequences include the kappa and lambda constant lightsequences. Preferred human heavy constant sequences include human gamma1, human gamma 2, human gamma 3, human gamma 4, and mutated versionsthereof which provide for altered effect or function, e.g., enhanced invivo half-life or reduced Fc receptor binding.

[0069] After expression, the antigen binding affinity of the resultinghumanized antibody will be assayed by known methods, e.g., Scatchardanalysis. In a particularly preferred embodiment, the antigen-bindingaffinity of the humanized antibody will be at least 25% of that of theparent antibody, e.g., CC49, i.e. a minimum of two-fold less than thatof native or chimeric CC49. Most preferably, the affinity of thehumanized antibody will be at least about 50% of that of the parentantibody, e.g., CC49.

[0070] In some instances, humanized antibodies produced by grafting CDRs(from an antibody which binds TAG-72) onto selected human frameworkregions may provide humanized antibodies having the desired affinity toTAG-72. However, it may be necessary or desirable to further modifyspecific residues of the selected human framework in order to enhanceantigen binding. This may occur because it is believed that someframework residues are essential to or at least affect antigen binding.Preferably, those framework residues of the parent (e.g., murine)antibody which maintain or affect combining-site structures will beretained. These residues may be identified by X-ray crystallography ofthe parent antibody or Fab fragment, thereby identifying thethree-dimensional structure of the antigen-binding site. Also, frameworkresidues involved in antigen binding may potentially be identified basedon previously reported humanized murine antibody sequences. Thus, it maybe beneficial to retain such framework residues or others from theparent murine antibody to optimize TAG-72 binding. Preferably, suchmethodology will confer a “human-like” character to the resultanthumanized antibody thus rendering it less immunogenic while retainingthe interior and contacting residues which affect antigen-binding.

[0071] The present invention further embraces variants and equivalentswhich are substantially homologous to the humanized antibodies andantibody fragments set forth herein. These may contain, e.g.,conservative substitution mutations, i.e. the substitution of one ormore amino acids by similar amino acids. For example, conservativesubstitution refers to the substitution of an amino acid with anotherwithin the same general class, e.g., one acidic amino acid with anotheracidic amino acid, one basic amino acid with another basic amino acid,or one neutral amino acid by another neutral amino acid. What isintended by a conservative amino acid substitution is well known in theart.

[0072] The phrase “substantially homologous” is used in regard to thesimilarity of a subject amino acid sequence (of an oligo- orpoly-peptide or protein) to a related, reference amino acid sequence.This phrase is defined as at least about 75% Correspondence”—i.e. thestate of identical amino acid residues being situated inparallel—between the subject and reference sequences when thosesequences are in “alignment,” i.e. when a minimal number of “null” baseshave been inserted in the subject and/or reference sequences so as tomaximize the number of existing bases in correspondence between thesequences. “Null” bases are not part of the subject and referencesequences; also, the minimal number of “null” bases inserted in thesubject sequence may differ from the minimal number inserted in thereference sequence. In this definition, a reference sequence isconsidered “related” to a subject sequence where both amino acidsequences make up proteins or portions of proteins which are eitherαTAG-72 antibodies or antibody fragments with αTAG-72 binding affinity.Each of the proteins comprising these αTAG-72 antibodies or antibodyfragments may independently be antibodies or antibody fragments or bi-or multi-functional proteins, e.g., such as fusion proteins, bi- andmulti-specific antibodies, single chain antibodies, and the like.

[0073] The present invention is further directed to nucleic acidsequences from such humanized antibodies may be expressed, as well asexpression vectors which provide for the production of such humanizedantibodies in transformed host cells.

[0074] In the preferred embodiments, such humanized antibodies andcorresponding nucleic acid sequences will be derived from CC49. Mostpreferably, the humanized heavy chains will have the amino acidsequences set forth in FIG. 1 or 3 and the humanized light chains willhave the amino acid sequences set forth in FIG. 2 or 4. However, asdiscussed, the invention further contemplates other modifications ofthese humanized variable heavy and light sequences, e.g., sequenceswhich further comprise one or more conservative amino acid substitutionsor sequences which retain one or more additional murine frameworkresidues which affect (enhance) antigen binding, which are alternativesto or supplements for those already shown in these Figures.

[0075] The subject humanized antibodies, because they specifically bindTAG-72, a pancarcinoma antigen expressed on many different cancer celltypes (e.g., colon carcinoma, breast carcinoma, ovarian carcinoma,prostate carcinoma), and further because they are expected to besignificantly non-immunogenic in humans, should be suitable for use astherapeutics for the treatment or prevention of cancers characterized byTAG-72 expression, and as diagnostic agents, e.g., for use in tumorimaging or in the Radioimmunoguided Surgery System (RIGS®). See Hinkleet al, Antibody, Immunoconjugates and Radiopharmaceuticals, 4(3):339-358(1991). One skilled in the art would be able, by routineexperimentation, to determine what an effective, non-toxic amount ofantibody would be for the purpose of treating cancer. Generally,however, an effective dosage will be in the range of about 0.05 to 100milligrams per kilogram body weight per day.

[0076] The antibodies of the invention may be administered to a mammalin accordance with the aforementioned methods of treatment in an amountsufficient to produce such effect to a therapeutic, prophylactic, ordiagnostic effect. Such antibodies of the invention can be administeredto such mammal in a conventional dosage form prepared by combining theantibody of the invention with a conventional pharmaceuticallyacceptable carrier or vehicle, diluent, and/or excipient according toknown techniques to form a suspension, injectable solution, or otherformulation. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

[0077] Pharmaceutically acceptable formulations may include, e.g., asuitable solvent, preservatives such as benzyl alcohol if desired, and abuffer. Useful solvent may include, e.g., water, aqueous alcohols,glycols, and phsophonate and carbonate esters. Such aqueous solutionscontain no more than 50% by volume of organic solvent. Suspension-typeformulations may include a liquid suspending medium as a carrier, e.g.,aqueous polyvinylpyrrolidone, inert oils such as vegetable oils orhighly refined mineral oils, or aqueous cellulose ethers such as aqueouscarboxymethylcellulose. A thickener such as gelatin or an alginate mayalso be present, one or more natural or synthetic surfactants orantifoam agents may be used, and one or more suspending agents such assorbitol or another sugar may be employed therein. Such formations maycontain one or more adjuvants.

[0078] The route of administration of the antibody (or fragment thereof)of the invention may be oral, parenteral, by inhalation or topical. Theterm parenteral as used herein includes intravenous, intramuscular,subcutaneous, rectal, vaginal or intraperitoneal administration. Thesubcutaneous, intravenous and intramuscular forms of parenteraladministration are generally preferred. The daily parenteral and oraldosage regimens for employing humanized antibodies of the inventionprophylactically or therapeutically will generally be in the range ofabout 0.005 to 100, but preferably about 0.5 to 10, milligrams perkilogram body weight per day.

[0079] The antibody of the invention may also be administered byinhalation. By “inhalation” is meant intranasal and oral inhalationadministration. Appropriate dosage forms for such administration, suchas an aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques. The preferred dosage amount of a compound ofthe invention to be employed is generally within the range of about 0.1to 1000 milligrams, preferably about 10 to 100 milligrams/kilogram bodyweight.

[0080] The antibody of the invention may also be administered topically.By topical administration is meant non-systemic administration. Thisincludes the administration of a humanized antibody (or humanizedantibody fragment) formulation of the invention externally to theepidermis or to the buccal cavity, and instillation of such an antibodyinto the ear, eye, or nose, and wherever it does not significantly enterthe bloodstream. By systemic administration is meant oral, intravenous,intraperitoneal, subcutaneous, and intramuscular administration. Theamount of an antibody required for therapeutic, prophylactic, ordiagnostic effect will, of course, vary with the antibody chosen, thenature and severity of the condition being treated and the animalundergoing treatment, and is ultimately at the discretion of thephysician. A suitable topical dose of an antibody of the invention willgenerally be within the range of about 1 to 100 milligrams per kilogrambody weight daily.

[0081] Formulations

[0082] While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation. The topical formulations of the presentinvention, comprise an active ingredient together with one or moreacceptable carrier(s) therefor and optionally any other therapeuticingredients(s). The carrier(s) must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

[0083] Formulations suitable for topical administration include liquidor semi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear, or nose. Drops according to the present invention maycomprise sterile aqueous or oily solutions or suspensions and may beprepared by dissolving the active ingredient in a suitable aqueoussolution of a bactericidal and/or fungicidal agent and/or any othersuitable preservative, and preferably including a surface active agent.The resulting solution may then be clarified and sterilized byfiltration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

[0084] Lotions according to the present invention include those suitablefor application to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

[0085] Creams, ointments or pastes according to the present inventionare semi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

[0086] Kits according to the present invention include frozen orlyophilized humanized antibodies or humanized antibody fragments to bereconstituted, respectively, by thawing (optionally followed by furtherdilution) or by suspension in a (preferably buffered) liquid vehicle.The kits may also include buffer and/or excipient solutions (in liquidor frozen form)—or buffer and/or excipient powder preparations to bereconstituted with water—for the purpose of mixing with the humanizedantibodies or humanized antibody fragments to produce a formulationsuitable for administration. Thus, preferably the kits containing thehumanized antibodies or humanized antibody fragments are frozen,lyophilized, pre-diluted, or pre-mixed at such a concentration that theaddition of a predetermined amount of heat, of water, or of a solutionprovided in the kit will result in a formulation of sufficientconcentration and pH as to be effective for in vivo or in vitro use inthe treatment or diagnosis of cancer. Preferably, such a kit will alsocomprise instructions for reconstituting and using the humanizedantibody or humanized antibody fragment composition to treat or detectcancer. The kit may also comprise two or more component parts for thereconstituted active composition. For example, a second componentpart—in addition to the humanized antibodies or humanized antibodyfragments—may be bifunctional chelant, bifunctional chelate, or atherapeutic agent such as a radionuclide, which when mixed with thehumanized antibodies or humanized antibody fragments forms a conjugatedsystem therewith. The above-noted buffers, excipients, and othercomponent parts can be sold separately or together with the kit.

[0087] It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a humanized antibody orhumanized antibody fragment of the invention will be determined by thenature and extent of the condition being treated, the form, route andsite of administration, and the particular animal being treated, andthat such optima can be determined by conventional techniques. It willalso be appreciated by one of skill in the art that the optimal courseof treatment, i.e., the number of doses of an antibody or fragmentthereof of the invention given per day for a defined number of days, canbe ascertained by those skilled in the art using conventional course oftreatment determination tests.

[0088] The subject humanized antibodies may also be administered incombination with other anti-cancer agents, e.g., other antibodies ordrugs. Also, the subject humanized antibodies or fragments may bedirectly or indirectly attached to effector having therapeutic activity.Suitable effector moieties include by way of example cytokines (IL-2,TNF, interferons, colony stimulating factors, IL-1, etc.), cytotoxins(Pseudomonas exotoxin, ricin, abrin, etc.), radionuclides, such as ⁹⁰Y,¹³¹I, ^(99m)Tc, ¹¹¹In, ¹²⁵I, among others, drugs (methotrexate,daunorubicin, doxorubicin, etc.), immunomodulators, therapeutic enzymes(e.g., beta-galactosidase), anti-proliferative agents, etc. Theattachment of antibodies to desired effectors is well known. See, e.g.,U.S. Pat. No. 5,435,990 to Cheng et al. Moreover, bifunctional linkersfor facilitating such attachment are well known and widely available.Also, chelators (chelants and chelates) providing for attachment ofradionuclides are well known and available.

[0089] Alternatively, the subject humanized antibodies or fragmentsspecific to TAG-72 may be used as immunodiagnostic agents both in vivoand in vitro. A particularly preferred usage is for in vivo imaging ofcancer cell lesions which express TAG-72. The subject antibodies arepreferred because they should elicit no significant HAMA or allergicresponse. Thus, they may be used repeatedly to monitor the diseasestatus of a patient.

[0090] As noted above, another preferred application of the subjecthumanized antibodies or fragments thereof is in the RadioimmunoguidedSystems®. This technique, also known as the RIGS® System involves theintravenous administration of a radiolabeled monoclonal antibody or itsfragment prior to surgery. After allowing for tumor uptake and bloodclearance of radioactivity, the patient is taken to the operating roomwhere surgical exploration is effected with the aid of a hand-held gammaactivity probe, e.g., Neoprobe®1000. This helps the surgeon identify thetumor metastases and improve the complications of excision. The RIGS®system is advantageous because it allows for the detection of tumors nototherwise detectable by visual inspection and/or palpation. See, O'Dwyeret al, Arch. Surg., 121:1 391-1394 (1986). This technique is describedin detail in Hinkle et al, Antibody, Immunoconjugates andRadiopharmacouticals, 4:(3)339-358 (1991) (citing numerous referencesdescribing this technique). This reference also discloses the use ofthis technique with the CC49 monoclonal antibody itself. This techniqueis particularly useful for cancers of the colon, breast, pancreas, andovaries.

[0091] The subject humanized antibodies or humanized antibody fragmentsthereof radiolabeled with radionuclides which are suitable for in vivoadministration, e.g., iodine radionuclides such as ¹³¹I and ¹²⁵I; ¹¹¹Inand ^(99m)Tc are also suitable radiolabels.

[0092] The subject humanized antibodies may be used alone or incombination with other antibodies. Also, the subject humanizedantibodies may be prepared in the form of a diagnostically effectivecomposition. Generally, this will entail the incorporation ofdiagnostically acceptable carriers and excipients, and labels whichprovide for detection. Suitable labels include diagnostic radionuclides,enzymes, etc. Methods for using antibodies for tumor imaging are wellknown in the art.

[0093] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The invention will be further clarifiedby a consideration of the following examples, which are intended to bepurely exemplary of the present invention are thus to be construed asmerely illustrative examples and not limitations of the scope of thepresent invention in any way.

EXAMPLES

[0094] Materials and Methods

[0095] DNA Template Preparation

[0096] All recombination work was performed upon DNA sequences inplasmid M13 vectors. The source of the NEWM framework regions forproducing the initial humanized CC49 VH was an M13 constructbearing—between the M13 BamH I and Hind III sites—a DNA segment havingthe nucleotide sequence shown in FIG. 13. The source of REI frameworkregions for producing the initial humanized CC49 VL was an M13 constructbearing—between the M13 BamH I and Hind III sites—a DNA segment encodingthe REI amino acid sequence of FIG. 2.

[0097] When overlap-extension procedures were used to introducemutations into a given DNA sequence, double stranded M13 DNA wasutilized. In contrast, when extension-ligation procedures were usedinstead, the oligonucleotides were designed to anneal to only one of thetwo DNA strands. In this latter procedure, the M13 DNA was first treatedto substitute uridine for every thymidine base in the DNA, to produceuridinylated DNA. This was accomplished by transfecting the M13 plasmidDNA into competent cells lacking dUTPase and uracil glycosylase,normally RZ1032 cells (though CJ236 cells available from Bio-Rad ofHercules, Calif., are also suitable) by combining the followingingredients.

[0098] 1 μL of M13 plasmid DNA

[0099] 4 mL of LB broth

[0100] 40 μL of competent RZ1032 cells.

[0101] The culture was shaken for 5 hours at 37° C. and the resultingsingle-stranded plasmid DNA (ssDNA) was isolated and dissolved in 50 μLTris-EDTA buffer. The DNA was then treated with uracil glycosylase bymixing together:

[0102] 1 μL uridinylated ssDNA

[0103] 1 μL 10× glycosylase buffer

[0104] 1 U uracil glycosylase (Gibco BRL, Gathersburg, Md.)

[0105] 40 μL 25 mM MgCI2.

[0106] This mixture was then incubated at 37° C. for one hour and then6.6 μL 25 mM MgCI2 and 9.9 μL 1M NaOH. The mixture was then furtherincubated for 5 minutes at 37° C. and 16.5 μL of 0.6M HCI was then addedto neutralize the mixture. The DNA was then ethanol precipitated anddissolved in water.

[0107] M13 Oligonucleotide Primers

[0108] The following oligonucleotide primers were used throughout theprocess of preparing the humanized CC49 VHs and VLs exemplified below.10. 5′-CTAAAACGACGGCCAGT-3′; 11. 5′-AACAGCTATGACCATG-3′; 385.5′-GCGGGCCTCTTCGCTATTACGC-3′ and 391. 5′-CTCTCTCAGGGCCAGGCGGTGA-3′.

[0109] These primers are complementary to regions of the plasmid M13which are external both to the (NEWM or REI) target framework sequencesand to the BamH I site-to-Hind III site section of M13.

[0110] Murine Variable-Regions

[0111] In order to compare the antibody binding characteristics of theantibodies produced according to the examples set forth below,antibodies having a chimeric heavy chain (i.e. a heavy chain having amurine CC49 VH region and a human IgG1 constant region) and/or achimeric light chain (i.e. a light chain having a murine CC49 VL and ahuman κ constant region) were expressed. The source of these chimeric

[0112] chains was the ATCC-deposited cell line HB9884 (Budapest) whichexpresses a chimeric CC49 antibody having both chains. The heavy chainof this antibody was termed “MuVH” and the light chain thereof wastermed “MuVL.”

[0113] Oligonucleotide Phosphorylation Protocol

[0114] Mutating oligonucleotides used in non-overlap extensions werephosphorylated according to the procedure below. In a final volume of 25μL, the following ingredients were combined:

[0115] 10 pmol of each oligonucleotide,

[0116] 5 μL of a 5× polynucleotide kinase buffer, and

[0117] 5U of T4 polynucleotide kinase (Gibco BRL).

[0118] The phosphorylation reaction was started with the addition of theenzyme and allowed to proceed for one hour at 37° C.

[0119] Annealing Protocol for Non-Overlap Extension-Ligations

[0120] The annealing step for non-overlap extension-ligations involvedperforming one annealing in which all mutation-carrying oligonucleotidesand one primer oligonucleotide were annealed to a single stranded DNAtemplate in which all thymidine bases had been replaced with uridinebases. The mutating oligonucleotides were first phosphorylated accordingto the above oligonucleotide phosphorylation protocol. In a final volumeof 20 μL, the following ingredients were combined:

[0121] 1 pmol of each mutation-carrying phosphorylated oligonucleotide

[0122] 1 pmol of a primer oligonucleotide

[0123] 4 μL 5× annealing buffer

[0124] 0.2 pmol ssU-DNA template.

[0125] The mixture was then heated to 90° C. for 30 sec., then quicklycooled to 70° C., and finally allowed to slowly cool to 37° C.

[0126] Extension-Ligation Protocol for Non-Overlap Extension-Ligations

[0127] After completion of the annealing step in which the primer andphosphorylated mutating oligonucleotides were annealed to the ssU-DNAtemplate, extension-ligation was performed as follows. In a final volumeof 30 μL, the following ingredients were combined:

[0128] 20 μL annealed ssU-DNA (i.e. the contents of the above annealingprocedure)

[0129] 2 μL 5× annealing buffer

[0130] 2 μL 0.1 M dithiothreitol

[0131] 0.3 μL 0.1M ATP

[0132] 1 μL 6.25 mM dNTP mixture of equimolar amounts of dATP, dTTP,dGTP, dCTP

[0133] 2.5U T7 DNA polymerase (USB, now Amersham Life Sciences,Cleveland, Ohio)

[0134] 0.5U T4 DNA ligase (Gibco BRL)

[0135] Water to 30 μL.

[0136] This mixture was then incubated at room temperature for 1 hour.

[0137] Standard PCR Protocols

[0138] The following procedure was used, alternately, both to amplifythe non-overlap extension-ligation DNA sequences and to performextension of each overlap DNA sequence. In a final volume of 50 μL, thefollowing ingredients were combined:

[0139] 2 μL template DNA (either annealed ssU-DNA or non-annealed ssDNA)

[0140] 5 μL 10× Vent buffer (NEB, i.e. New England Biolabs, Beverly,Mass.) or 10× Thermalase buffer (IBI of New Haven, Conn.)

[0141] 2 μL 6.25 mM dNTP mixture of equimolar amounts of dATP, dTTP,dGTP, dCTP

[0142] 25 pmol of one oligonucleotide primer

[0143] 25 pmol of either a mutation-carrying oligonucleotide (foroverlap-extension) or a second oligonucleotide primer

[0144] 1 U Vent DNA polymerase (NEB) or Thermalase DNA polymerase (IBI).

[0145] Reactions were initiated with the addition of the DNA polymeraseand then treated with about 15 cycles of: (1) 94° C. for 30 sec., (2)50° C. for 30 sec., and (3) 30-60 seconds at either 75° C. (for Vent DNApolymerase) or 72° C. (for Thermalase). Reactions were brought tocompletion with 5 minutes at a constant temperature of either 75° C. ForVent DNA polymerase) or 72° C. (for Thermalase).

[0146] PCR Overlap-Extension Amplification Protocol

[0147] After a pair of PCR reactions were performed—one for each of thetwo (partially complementary) overlapping DNA segments—the two resultingsegments were joined according to the following PCR procedure. In afinal volume of 50 μL, the following ingredients were mixed:

[0148] 1 μL of each overlap DNA (from the above overlap PCR extensionreactions)

[0149] 5 μL 10× Vent buffer (NEB) or Thermalase buffer (IBI)

[0150] 2 μL 6.25 mM dNTP mixture of equimolar amounts of dATP, dTTP,dGTP, dCTP

[0151] 25 pmol of each oligonucleotide primer used in the overlap PCRextensions

[0152] 1 U Vent DNA polymerase (NEB) or Thermalase DNA polymerase (IBI).

[0153] Reactions were initiated with the addition of the DNA polymeraseand then treated with about 15 cycles of: (1) 94° C. for 30 sec., (2)50° C for 30 sec., and (3) 30-60 seconds at either 75° C. (for Vent DNApolymerase) or 72° C. (for Thermalase). Reactions were brought tocompletion with 5 minutes at a constant temperature of either 75° C.(for Vent DNA polymerase) or 72° C. (for Thermalase).

[0154] Transfer of Humanized CC49 Variable Region DNA Sequences from M13to pSV Vectors and Subsequent Antibody Expression

[0155] Humanized antibodies were expressed in pSV vectors grown in NSOcells. The humanized variable region constructs which were produced inthe plasmid, M13, were digested with 10U each of Hind III and BamH I(both from BRL, i.e. Gibco BRL) for 1 hour at 37° C. in a final volumeof 100 μL with Tris-EDTA buffer. The resulting DNA fragments were thenrun on a low melting point agarose gel, the band containing thehumanized construct DNA was cut out, and the DNA was purified using anELUTIP ‘d’ column with 20 μL Tris-EDTA buffer. 10 μL of the purified DNApreparation was then combined with 1 μL of a Hind III and BamH1-digested pSV preparation, 3 μL of 5× ligase buffer, and 1U of T4 DNAligase (BRL), in order to insert the construct into a pSV plasmid.Humanized CC49 VH constructs were inserted into pSVgpt vectors bearing ahuman IgG1 heavy chain constant region; the pSVgpt vector used is the“aLYS-30” shown in FIG. 5. Humanized CC49 VL constructs were insertedinto pSVhyg vectors bearing a human K light chain constant domain; thepSVhyg vector used in the “aLys-17” shown in FIG. 5. Each humanizedvariable region construct was inserted adjacent to the respectiveconstant region, i.e. so as to replace either the HuVHLYS or the HuVLLyssegment illustrated in FIG. 5.

[0156] The resulting vectors were transfected into NSO cells as follows.About 3 μg of the VH vector, or about 6 μg of the VL vector, produced bythe pSV-insertion procedures, was then linearized by digestion with 10 UPvul (Gibco BRL). The digested DNA was then precipitated with ethanoland redissolved in 50 μL of water. NSO cells were collected bycentrifugation and resuspended in 0.5 mL Dulbecco's Modified Eagle'sMedium (DMEM) and then transferred to a Gene Pulser cuvette (Bio-Rad).The DNA from both one VH and one VL construct was gently mixed with thecells by pipetting and the cuvette was left on ice for 5 minutes. Next,the cuvette was inserted between the electrodes of the Bio-Rad GenePulser and a single pulse of 170V at 960 μF was applied. The contents ofthe cuvette were then transferred to a flask containing 20 mL DMEM andthe cells were allowed to rest for 1-2 days at 37° C. Cells were againharvested by centrifugation and resuspended in 36 mL selective DMEM. 1.5mL aliquots of this resuspension were placed in each well of a 24-wellplate and incubated at 37° C. for 4 days, at which time the medium ineach well was replaced with 1.5 mL of fresh selective DMEM. After 6 moredays of incubation at 37° C., surviving cell colonies were visible tothe naked eye and the supernatants of each well were assayed forantibody production. Both whole antibody production (i.e. withoutpurification) and purified antibody production were assayed. To obtainpurified antibodies, the supernatants were passed through a protein Acolumn.

[0157] ELISA Assay Protocols

[0158] Antibody concentrations and antibody binding characteristics weretested using enzyme-linked immunosorbent assay (ELISA) procedures whichare set forth as follows.

[0159] Measurement of IgG concentration

[0160] The concentration of IgG secreted from transfected cells wasmeasured using an enzyme-linked immunosorbent assay (ELISA) procedurewhich is set forth as follows.

[0161] Polyvinyl chloride (PVC) microtiter plates (DynatechLaboratories, Chantilly, Va., catalog # 001-010-2101) were coated withgoat anti-human IgG (10 mg/mL, GAHIG, Southern Biotechnology Associates,Inc., Birmingham, Ala., catalog # 2010-01) diluted with Milli-Q® waterand placed on the plates using 50 mL/well. Plates were air-driedovernight at ambient temperature or at 37° C. for 3 hours. Prior to use,non-specific binding was blocked the addition of 0.2 mL/well of 1% (w/v)bovine serum albumin (Sigma, St. Louis, Mo. catalog # A7888) inphosphate buffered saline (Sigma, catalog # 1000-3) (PBS/BSA). Allincubations were carried out in a humidified container. Plates wereincubated for 1-2 hours at 37° C. and the blocking solution removedprior to sample addition. Two-fold serial dilutions of samples or astandard IgG solution set at 500 ng/mL (50 μL/well) were made intriplicate in the PBS/BSA solution. The plate was incubated at 37° C.for 3 hours or overnight at 4° C. The plate was washed 3 times with0.025% Tween-20 (v/v, Sigma) using an automatic plate washer. 50 μL/wellof 1:1000 dilution of a goat anti-human IgG conjugated to HorseradishPeroxidase (Southern Biotechnology Associates Inc.) was added andincubated at 37° C. for 1.5 hours. The wells were washed 3 times with0.025% Tween-20 (v/v, Sigma) using an automatic plate washer and 50μL/well OPD substrate buffer added. The color was developed for 4minutes, stopped with 12.5 μL 12.5% H₂SO₄ and the absorbance at 492 nmread. The concentration of IgG in the test sample was estimated bycomparison of the mean of the optical densities to a standard curveconstructed from the standard IgG.

[0162] Determination of Relative Affinities of Humanized Antibodies

[0163] Antibody binding characteristics were tested in an ELISA usingpartially purified TAG-72 antigen immobilized on Polyvinyl chloride(PVC) microtiter plates (Dynatech Laboratories, Chantilly, Va., catalog# 001-010-2101)

[0164] PVC plates were coated with 50 μL/well TAG-72 (Dow Chemical, lot#040191), diluted 1:300 in Milli-Q water. Plates were air-driedovernight at ambient temperature or at 37° C. for 3 hours. Prior to use,non-specific binding was blocked the addition of 0.2 mL/well of 1% (w/v)bovine serum albumin (Sigma, St. Louis, Mo. catalog # A7888) inphosphate buffered saline (Sigma, catalog # 1000-3) (PBS/BSA). Plateswere incubated for 1-2 hours at 37° C. and the blocking solution removedprior to sample addition. All incubations were carried out in ahumidified container. Two-fold serial dilutions (starting concentrationrange of 1.0 μg/ml-10 μg/ml) of the samples to be tested in the PBS/BSAsolution were added to triplicate wells of the TAG-coated plate (50μL/well). The plate was incubated overnight at 4° C. or 1-2 hours at 37°C. The plate was washed 3-times with 0.025% Tween-20 (v/v, Sigma) usingan automatic plate washer. 50 μL/well of 1:1000 dilution of a goatanti-human IgG conjugated to Horseradish Peroxidase (SouthernBiotechnology Associates Inc.) was added and incubated at 37° C. for 1.5hours. The wells were washed 3 times with 0.025% Tween-20 (v/v, Sigma)using an automatic plate washer and 50 μL/well OPD substrate bufferadded. The color was developed for 4 minutes, stopped with 12.5 μL 12.5%H₂SO₄ and the absorbance at 492 nm read.

[0165] Determination of Affinity Constants for Binding to TAG-72

[0166] Two-fold dilutions of purified Hu-CC49 were prepared in PBS/BSAover a range of 1.0 μg/ml-0.003 μg/ml and samples (20 μL/well) wereapplied in triplicate to TAG coated PVC prepared and blocked asdescribed supra. Plates were incubated overnight at 4° C. Following thisincubation, samples were transferred from the plate to the correspondingwells on the GAHIG-coated trap plate. The original TAG plate was washed3-times with 0.025% Tween-20 (v/v, Sigma, catalog # P1379) using anautomatic plate washer. An ¹²⁵I-labeled goat anti-human IgG probe (ICNBiomedicals, Inc., catalog # 68088) was diluted to 75,000 cpm/25 μL inPBS/BSA and added (25 μL/well) to all wells. This TAG plate wasincubated for 1 hour at 37° C.

[0167] After a 1 hour incubation at 37° C., the trap plate was washed asdescribed above and ¹²⁵I-labeled GAHIG probe was added. This plate wasincubated for 1 hour at 37° C. Both plates (TAG and GAHIG-trap)containing probe were then washed with a microplate washer to remove theunbound probe. A plate cutter (D. Lee, Sunnyvale, Calif., Model HWC-4)was used to separate the wells from the plate frame. The radioactivityin each well was quantified by a gamma counter. The resulting data wasanalyzed according to the method of Scatchard (Ann. NY Acad., 51:600-672(1946)).

EXAMPLE 1

[0168] Preparation of CDR-grafted (Initial Humanized) Antibody fromMurine CC49

[0169] We describe in this Example the construction of humanized CC49Mabs (CC49 HuVH/HuVK) using the V_(L) and V_(H) frameworks of human MabsREI and NEWM, respectively. The CDRs for murine CC49 were grafted ontohuman frameworks according to known methods as discussed supra. Inparticular, human frameworks were selected from antibodies which, basedon previous studies, were predicted to be suitable, i.e. which shouldnot adversely affect antigen binding and not exhibit significantimmunogenicity in humans. The human frameworks selected for the variableheavy and variable light chains, respectively, were NEWM and REI. In theproduction of the initial version of the humanized VH, certain murineframework residues were also retained which, based on previous studies,might allow retention of antigen binding properties. Specifically,residues Y27, T30, A72, F95, and T97 of the murine heavy chain wereinitially retained. Concurrently, an alternate version of the humanizedVH was produced which retained, in addition, the murine frameworkresidue A24.

[0170] The production of these NEWM-grafted humanized CC49 VHS wasaccomplished according to the annealing and extension-ligation protocolsdescribed above, using a single-stranded M13 DNA template bearing,between the Hind III and BamH I sites thereof, a DNA segment having thenucleotide sequence shown in FIG. 13. In this procedure, Primer 11 wasused in conjunction with a set of mutating oligonucleotides. Thesemutating oligonucleotides were designed and synthesized with thefollowing sequences: 1a.5′GCTGTCTCACCCAGTGAATTGCATGGTCAGTGAAGGTGTAGCCAGA CACGGTGCAGGTCA-3′; 1b.5′GCTGTCTCACCCAGTGAATTGCATGGTCAGTGAAGGTGTAGCCAGA CGCGGTGCAGGTCA-3′; 2.5′CTGGTGTCTGCCAGCATTGTCACTCTCCCCTTGAACCTCTCATTGTATTTAAAATCATCATTTCCGGGAGAAAAATATCCAATCCACTCAAGAC-3′; and 3.5′GGACCCTTGGCCCCAGTAGGCCATATTCAGGGATCTTGTACAGAAAT AGACCGCGGTGTC-3′.

[0171] Codons which were designed into the oligonucleotides in order toretain murine FR amino acids are shown in bold-face type. Afterextension-ligation, amplifying PCR was performed using the standard PCRprotocol with Vent DNA polymerase. The use of two versions of mutatingoligonucleotide 1 resulted in the formation of two initial humanizedV_(H)S. These were named “CC49 NMVH,” also called “HuVH,” (forconstructs incorporating oligonucleotide 1) and “HuVHA” (for constructsincorporating oligonucleotide 1b).

[0172] In the production of the initial version of the humanized VL, nouniquely murine framework residues were retained. The production of theREI-grafted humanized CC49 V, was accomplished according to theannealing and extension-ligation protocols described above, using assM13 template bearing, between the Hind III and BamH I sites thereof, assU-DNA segment encoding the REIVK sequence shown in FIG. 2. In thisprocedure, Primer 385 was used in conjunction with a set of mutatingoligonucleotides. These mutating oligonucleotides were designed andsynthesized with the following sequences: 21.5′-GTTCTTCTGATTACCACTGTATAAAAGACTTTGACTGGAC-3′; 22.5′-CAGATTCCCTAGCGGATGCCCAGTAG-3′; 23.5′-TTCTACTCACGTGTGATTTGCAGCTTGGTCCCTTGGCCGAACGTGAGGGGATAGGAATAGTATTGCTGGCAGTAGTAG-3′; and 24.5′-GCTCTGGGTCATCTGGATGTCGG-3′.

[0173] After extension-ligation, amplifying PCR was performed using theThermalase standard PCR protocol described above. The resultinghumanized V_(L) was named “CC49 REVK” and was also called “HuVK.”

[0174] The two heavy chain constructs, HuVH and HuVHA, and the lightchain construct, HuVH, which were situated in Ml 3 vectors, were grownand expressed in TG1 cells. The polypeptide expression products of theseconstructs were sequenced and the amino acid sequences of theseconstructs are presented in FIGS. 1 and 2.

[0175] These DNA constructs were then inserted into pSV expressionvectors as described above. Combinations of these with each other orwith the ATCC (Budapest) HB 9884 DNA sequences encoding the chimericMuVH or MuVL were then inserted into NSO cells. Specifically, thefollowing four combinations of heavy and light chain constructs wereseparately transfected into NSO cells as described above: HuVHA andMuVK, HuVHA and HuVK, MuVH and MuVK, and HuVH and HuVK. Thesecombinations were expressed and the resulting antibodies were thentested for antigen binding characteristics using the ELISA assay setforth above. The results of these assays are shown in FIGS. 6-9.

[0176] The data in FIG. 6 show that, with HuVHA, the HuVK humanizedlight chain functions as well as the MuVK chimeric light chain. FIGS. 7and 8 indicate that the fully humanized HuVH/HuVK antibody binds TAG-72approximately 2-fold less than the chimeric MuVH/MuVK antibody. Also,FIG. 9 suggests that the A24 mutation produces no enhancement in antigenbinding; rather, the A24 mutation causes an approximate 2-fold reductionin affinity as measured by this ELISA assay.

[0177] Further Development of Humanized CC49

[0178] Because it appears that the HuVK humanized light chain functionsas well as the MuVK chimeric light chain, further work was directed tomaking alternate mutated versions of the HuVH humanized heavy chain. Afirst variant of HuVH was made by replacing the lysine residue shown atposition 76 of the CC49 NMVH in FIG. 1 with the murine FR residue,serine. This was achieved by the Vent DNA polymerase PCR overlapextension protocol described above, using two primer-and-mutatingoligonucleotide pairs—oligonucleotides 10 and 4, and oligonucleotides 11and 5. Each pair was used in conjunction with one of the strands of adsDNA template—situated between the Hind III and BamH I sites of plasmidM13—and having the nucleotide sequence shown in FIG. 14 (the upperstrand was used with pair 11 & 5). Mutating oligonucleotides 4 and 5were designed and synthesized with the following sequences: 4.5′-AGACACCAGCAGCAACCAGTTCAG-3′; and 5. 5′-GCTGAACTGGTTGCTGCTGGTGTC-3′.

[0179] The serine residue codons are shown in bold-face type. Theresulting humanized CC49 V_(H) was named “HuVHS.”

[0180] A second variant of HUVH was made by replacing the threonineresidue shown at position 74 of the CC49 NMVH in FIG. 1 with the murineFR residue, lysine. This was achieved by the Vent DNA polymerase PCRoverlap extension protocol described above, using twoprimer-and-mutating oligonucleotide pairs—oligonucleotides 10 and 6, andoligonucleotides 11 and 7. Each pair was used in conjunction with one ofthe strands of a dsDNA template—situated between the Hind III and BamH Isites of plasmid M13—and having the nucleotide sequence shown in FIG. 14(the upper strand was used with pair 11 & 7). Mutating oligonucleotides6 and 7 were designed and synthesized with the following sequences. 6.5′-CTGGCAGACAAGAGCAAGAACCAG-3′. 7. 5′-TGGTTCTTGCTCTTGTCTGCCAGC-3′.

[0181] The lysine residue codons are shown in bold-face type. Theresulting humanized CC49 VH was named “HuVHK.”

[0182] The two heavy chain constructs, HUVHS and HUVHK, which weresituated in M13 vectors, were grown and expressed in TG1 cells. Thepolypeptide expression products of these constructs were sequenced andthe amino acid sequences of these constructs are presented in FIG. 1.

[0183] These DNA constructs were then inserted into pSV expressionvectors as described above. Combinations of these with HUVK were theninserted into NSO cells. These combinations were expressed and theresulting antibodies were then tested for antigen bindingcharacteristics using the ELISA assay set forth above. The results ofthese assays are shown in FIGS. 10 and 11. These figures show thatneither the K74 nor S76 mutation resulted in enhanced antigen binding;in fact the S76 mutation caused an approximate 2-fold reduction inaffinity as measured by this ELISA assay.

EXAMPLE 2

[0184] Measurement of Affinity Constant for the Humanized CC49 AntibodyObtained in Example 1

[0185] The affinity constant of the final humanized CC49 antibody, CC49HuVH/HuVK, of Example 1 (having variable heavy and variable light chainsequences shown in FIGS. 3 and 4, respectively) were measured accordingto the ELISA assay protocol set forth above. The ATCC (Budapest) HB 9884chimeric CC49 antibody was used as an internal control. This ELISA assaywas performed repeatedly using purified samples of humanized andchimeric CC49 monoclonal antibodies in order to substantiate theaccuracy of the values obtained and to account for inherentassay-to-assay variability. Typical results from such an analysis areshown in FIG. 12. A summary of the results obtained during theseanalyses is shown in Table 1. TABLE 1 Summary of Affinity ConstantAnalysis of Humanized & Chimeric CC49 Chimeric CC49¹ CC49 HuVH/HuVKAffinity Constant (Ka) 7.62 × 10⁹M⁻¹ 4.27 × 10⁹M⁻¹ ± standard deviation3.94 × 10⁹M⁻¹ 2.57 × 10⁹M⁻¹ number of analyses 7 8

[0186] These results demonstrate that the humanized anti-TAG-72 antibodyHuVH/HuVK (having the variable heavy and variable light sequences shownin FIGS. 3 and 4, respectively) has a binding affinity approximatingthat of a chimeric CC49 antibody (MuVH/MuVK). Therefore, it is expectedthat this humanized antibody will effectively target TAG-72-expressingcarcinomas in vivo. Also, based on its sequence this antibody shouldexhibit little or no immunogenicity in humans, and exhibit advantageousplasma clearance, metabolic properties, and effective tumor targeting inrelation to the murine CC49, and also in relation to chimeric versionsthereof.

[0187] A murine plasmacytoma cell line which produces this humanizedCC49 antibody was deposited with the American Type Culture Collection(12301 Parklawn Drive, Rockville, Md. 20852, on Oct. 16, 1996) and thiscell line was accorded accession number ATCC CRL-12209. This deposit wasmade in accordance with the Budapest Treaty. This deposited cell linewill be made irrevocably available, without restriction, upon issuanceof a patent to this application or any other application claimingpriority to this application under 35 U.S.C. § 120.

[0188] Based on the foregoing, it will be appreciated that the humanizedantibodies disclosed in Examples 1-3, exhibit antigen-bindingcharacteristics, i.e. TAG-72 affinities comparable to the parentmonoclonal antibody, nCC49 (murine antibody), and to chimeric antibodiesderived from nCC49, e.g., cCC49. Moreover, based on the foregoingresults, these antibodies possess properties which will render them wellsuited for usage as in vivo diagnostics or therapeutics, e.g., improvedserum clearance, metabolic properties, and little or no immunogenicityin humans.

[0189] These properties are highly significant because these propertieswill enable the subject humanized antibodies to be administeredrepeatedly, in large dosages, and over a prolonged period of timewithout significant adverse effects, e.g., a HAMA response ornon-specific cytotoxicity. This is important for cancer treatment aswell as for cancer diagnosis as it enables these antibodies to be usedover prolonged time periods. Moreover, the clearance properties of thesubject human antibodies will enable these antibodies to effectivelytarget desired target sites, e.g., TAG-72 expressing carcinomas (becauseof the effects of serum clearance on targeting efficiency). Therefore,the humanized antibodies of the present invention comprise a substantialimprovement in relation to previously disclosed antibodies specific toTAG-72.

[0190] Other embodiments of the invention will be apparent to thoseskilled in the art from a consideration of this specification orpractice of the invention disclosed herein. It is intended that thespecification and examples be considered as exemplary only, with thetrue scope and spirit of the invention being indicated by the followingclaims.

1 33 1 115 PRT Mus musculus Murine CC49 VH 1..115 1 Gln Val Gln Leu GlnGln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 5 10 15 Ser Val Lys Ile SerCys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp ValLys Gln Asn Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser ProGly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Lys Ala ThrLeu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Val Gln Leu AsnSer Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser LeuAsn Met Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser115 2 115 PRT Homo sapiens NEWM VH FR template 1..115 CDR amino acidsare indicated by Xaa 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu ValArg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Trp Val Arg Gln Pro Pro Gly Arg GlyLeu Glu Trp Ile 35 40 45 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Thr Ser LysAsn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly GlnGly Ser Leu Val Thr 100 105 110 Val Ser Ser 115 3 115 PRT ArtificialSequence Humanized CC49 VH, HuVH 1..115 Heavy chain variable regioncontaining human NEWM FRs and murine CC49 VH CDRs 3 Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 5 10 15 Thr Leu Ser Leu ThrCys Thr Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp ValArg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser ProGly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val ThrMet Leu Ala Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu SerSer Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser LeuAsn Met Ala Tyr Trp Gly Gln Gly Ser Leu Val Thr 100 105 110 Val Ser Ser115 4 115 PRT Artificial Sequence Humanized CC49 VH, HuVHA 1..115 Heavychain variable region containing human NEWM FRs and murine CC49 VH CDRs,and retaining a murine alanine in FR1 4 Gln Val Gln Leu Gln Glu Ser GlyPro Gly Leu Val Arg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys Thr AlaSer Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Arg Gln ProPro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn AspAsp Phe Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Met Leu AlaAsp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val ThrAla Ala Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser Leu Asn Met AlaTyr Trp Gly Gln Gly Ser Leu Val Thr 100 105 110 Val Ser Ser 115 5 115PRT Artificial Sequence Humanized CC49 VH, HuVHS 1..115 Heavy chainvariable region containing human NEWM FRs and murine CC49 VH CDRs, andretaining a murine serine in FR3 5 Gln Val Gln Leu Gln Glu Ser Gly ProGly Leu Val Arg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val SerGly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp Val Arg Gln Pro ProGly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp AspPhe Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Met Leu Ala AspThr Ser Ser Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr AlaAla Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala TyrTrp Gly Gln Gly Ser Leu Val Thr 100 105 110 Val Ser Ser 115 6 115 PRTArtificial Sequence Humanized CC49 VH, HuVHK 1..115 Heavy chain variableregion containing human NEWM FRs and murine CC49 VH CDRs, and retaininga murine lysine in FR3 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu ValArg Pro Ser Gln 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr ThrPhe Thr Asp His 20 25 30 Ala Ile His Trp Val Arg Gln Pro Pro Gly Arg GlyLeu Glu Trp Ile 35 40 45 Gly Tyr Phe Ser Pro Gly Asn Asp Asp Phe Lys TyrAsn Glu Arg Phe 50 55 60 Lys Gly Arg Val Thr Met Leu Ala Asp Lys Ser LysAsn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp ThrAla Val Tyr Phe Cys 85 90 95 Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly GlnGly Ser Leu Val Thr 100 105 110 Val Ser Ser 115 7 113 PRT Mus musculusMurine CC49 VK 1..113 7 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu ProVal Ser Val Gly 5 10 15 Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln SerLeu Leu Tyr Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln GlnLys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala ArgGlu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Ser 65 70 75 80 Ile Ser Ser Val Lys Thr Glu Asp Leu Ala ValTyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala GlyThr Lys Leu Val Leu 100 105 110 Lys 8 113 PRT Homo sapiens REI VK FRtemplate 1..113 CDR amino acids are indicated by Xaa 8 Asp Ile Gln LeuThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15 Asp Arg Val ThrIle Thr Cys Lys Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa LysAsn Tyr Leu Ala Trp Tyr Gln Gln Thr Pro Gly Lys 35 40 45 Ala Pro Lys LeuLeu Ile Tyr Trp Ala Xaa Xaa Xaa Glu Ser Gly Val 50 55 60 Pro Ser Arg PheSer Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe THr 65 70 75 80 Ile Ser SerLeu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Xaa Xaa 85 90 95 Xaa Xaa XaaXaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Gln Ile 100 105 110 Thr 9113 PRT Artificial Sequence Humanized CC49 VK, HuVK 1..113 Light chainvariable region containing human REI FRs and murine CC49 VL CDRs 9 AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15 AspArg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 GlyAsn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Thr Pro Gly Lys 35 40 45 AlaPro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr 65 70 75 80Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile 100 105110 Thr 10 115 PRT Mus musculus Murine CC49 VH 1..115 10 Gln Val Gln LeuGln Gln Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 5 10 15 Ser Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His TrpVal Lys Gln Asn Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Phe SerPro Gly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Lys AlaThr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Val Gln LeuAsn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg SerLeu Asn Met Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val SerSer 115 11 115 PRT Artificial Sequence HuCC49 VH 1..115 Heavy chainvariable region containing human NEWM FRs and murine CC49 VH CDRs 11 GlnVal Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 5 10 15 ThrLeu Ser Leu Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 AlaIle His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 GlyTyr Phe Ser Pro Gly Asn Asp Asp Phe Lys Tyr Asn Glu Arg Phe 50 55 60 LysGly Arg Val Thr Met Leu Ala Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys 85 90 95Thr Arg Ser Leu Asn Met Ala Tyr Trp Gly Gln Gly Ser Leu Val Thr 100 105110 Val Ser Ser 115 12 117 PRT Homo sapiens NEWM VH 1..117 12 Gln ValGln Leu Glu Gln Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 5 10 15 Thr LeuSer Leu Thr Cys Thr Val Ser Gly Ser Thr Phe Ser Asn Asp 20 25 30 Tyr TyrThr Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly TyrVal Phe Tyr His Gly Thr Ser Asp Asp Thr Thr Pro Leu Arg 50 55 60 Ser ArgVal Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 ArgLeu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 ArgAsn Leu Ile Ala Gly Cys Ile Asp Val Trp Gly Gln Gly Ser Leu 100 105 110Val Thr Val Ser Ser 115 13 113 PRT Mus musculus Murine CC49 VL 1..113 13Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Pro Val Ser Val Gly 5 10 15Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser 65 70 7580 Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 9095 Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Val Leu 100105 110 Lys 14 113 PRT Artificial Sequence HuCC49 VL 1..113 Light chainvariable region containing human REI FRs and murine CC49 VL CDRs 14 AspIle Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15 AspArg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 GlyAsn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Thr Pro Gly Lys 35 40 45 AlaPro Lys Leu Leu Ile Tyr Trp Ala Ser Ala Arg Glu Ser Gly Val 50 55 60 ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr 65 70 75 80Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile 100 105110 Thr 15 107 PRT Homo sapiens REI VL 1..107 15 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15 Asp Arg Val Thr Ile ThrCys Gln Ala Ser Gln Asp Ile Ile Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln GlnThr Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn LeuGln Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr AspTyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala ThrTyr Tyr Cys Gln Gln Tyr Gln Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln GlyThr Lys Leu Gln Ile Thr 100 105 16 345 DNA Homo sapiens Template used toproduce HuVH and HuVHA 1..345 CDR amino acid-encoding codons areindicated by NNN 16 cag gtc caa ctg cag gag agc ggt cca ggt ctt gtg agacct agc cag 48 acc ctg agc ctg acc tgc acc gtg tct ggc nnn nnn nnn nnnnnn nnn 96 nnn nnn nnn tgg gtg aga cag cca cct gga cga ggt ctt gag tggatt 144 gga nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn nnn192 nnn nnn nnn nnn nnn nnn nnn nnn gac acc agc aag aac cag ttc agc 240ctg aga ctc agc agc gtg aca gcc gcc gac acc gcg gtc tat nnn nnn 288 nnnnnn nnn nnn nnn nnn nnn nnn tgg ggc caa ggg tcc ttg gtc acc 336 gtc tcctca 345 17 345 DNA Artificial Sequence Template used to produce HuVHSand HuVHK 1..345 DNA encoding a humanized heavy chain variable regioncontaining human NEWM FRs and murine CC49 VH CDRs 17 cag gtc caa ctg caggag agc ggt cca ggt ctt gtg aga cct agc cag 48 acc ctg agc ctg acc tgcacc gtg tct ggc tac acc ttc act gac cat 96 gca att cac tgg gtg aga cagcca cct gga cga ggt ctt gag tgg att 144 gga tat ttt tct ccc gga aat gatgat ttt aaa tac aat gag agg ttc 192 aag ggg aga gtg aca atg ctg gca gacacc agc aag aac cag ttc agc 240 ctg aga ctc agc agc gtg aca gcc gcc gacacc gcg gtc tat ttc tgt 288 aca aga tcc ctg aat atg gcc tac tgg ggc caaggg tcc ttg gtc acc 336 gtc tcc tca 345 18 17 DNA Artificial SequenceOligonucleotide 10 1..17 Oligonucleotide primer for plasmid M13 18ctaaaacgac ggccagt 17 19 16 DNA Artificial Sequence Oligonucleotide 111..16 Oligonucleotide primer for plasmid M13 19 aacagctatg accatg 16 2022 DNA Artificial Sequence Oligonucleotide 385 1..22 Oligonucleotideprimer for plasmid M13 20 gcgggcctct tcgctattac gc 22 21 22 DNAArtificial Sequence Oligonucleotide 391 1..22 Oligonucleotide primer forplasmid M13 21 ctctctcagg gccaggcggt ga 22 22 60 DNA Artificial SequenceOligonucleotide 1a 1..60 Mutagenic oligonucleotide for retaining 2murine FR amino acids in a humanized VH 22 gctgtctcac ccagtgaattgcatggtcag tgaaggtgta gccagacacg gtgcaggtca 60 23 60 DNA ArtificialSequence Oligonucleotide 1b 1..60 Mutagenic oligonucleotide forretaining 3 murine FR amino acids in a humanized VH 23 gctgtctcacccagtgaatt gcatggtcag tgaaggtgta gccagagcgg gtgcaggtca 60 24 94 DNAArtificial Sequence Oligonucleotide 2 1..94 Mutagenic oligonucleotidefor retaining 1 murine FR amino acid in a humanized VH 24 ctggtgtctgccagcattgt cactctcccc ttgaacctct cattgtattt aaaatcatca 60 tttccgggagaaaaatatcc aatccactca agac 94 25 60 DNA Artificial SequenceOligonucleotide 3 1..60 Mutagenic oligonucleotide for retaining 2 murineFR amino acids in a humanized VH 25 ggacccttgg ccccagtagg ccatattcagggatcttgta cagaaataga ccgcggtgtc 60 26 39 DNA Artificial SequenceOligonucleotide 21 1..39 Mutagenic oligonucleotide for retaining murineCDR amino acids in a humanized VL 26 gttcttctga ttaccactgt ataaaagacttgactggac 39 27 26 DNA Artificial Sequence Oligonucleotide 22 1..26Mutagenic oligonucleotide for retaining murine CDR amino acids in ahumanized VL 27 cagattccct agcggatgcc cagtag 26 28 78 DNA ArtificialSequence Oligonucleotide 23 1..78 Mutagenic oligonucleotide forretaining murine CDR amino acids in a humanized VL 28 ttctactcacgtgtgatttg cagcttggtc ccttggccga acgtgagggg ataggaatag 60 tattgctggcagtagtag 78 29 23 DNA Artificial Sequence 1..23 Mutagenicoligonucleotide for retaining murine CDR amino acids in a humanized VL29 gctctgggtc atctggatgt cgg 23 30 24 DNA Artificial SequenceOligonucleotide 4 1..24 Mutagenic oligonucleotide for retaining a murineserine in FR3 of HuVHS 30 agacaccagc agcaaccagt tcag 24 31 24 DNAArtificial Sequence Oligonucleotide 5 1..24 Mutagenic oligonucleotidefor retaining a murine serine in FR3 of HuVHS 31 gctgaactgg ttgctgctggtgtc 24 32 24 DNA Artificial Sequence Oligonucleotide 6 1..24 Mutagenicoligonucleotide for retaining a murine lysine in FR3 of HuVHK 32ctggcagaca agagcaagaa ccag 24 33 24 DNA Artificial SequenceOligonucleotide 7 1..24 Mutagenic oligonucleotide for retaining a murinelysine in FR3 of HuVHK 33 tggttcttgc tcttgtctgc cagc 24

What is claimed is:
 1. A humanized antibody or humanized antibodyfragment which specifically binds TAG-72 wherein said humanized antibodyor humanized antibody fragment is derived from a murine antibody thatbinds TAG-72.
 2. The humanized antibody or humanized antibody fragmentof claim 1 comprising CDRs which are obtained from said murine antibody,and comprising VH FRs which have an amino acid sequence of the NEWM FRsor the humanized CC49 FRs of FIG. 1 or 3, and comprising VL FRs whichhave an amino acid sequence of the REI FRs or the humanized CC49 FRs ofFIG. 2 or
 4. 3. The humanized antibody or humanized antibody fragment ofclaim 2 wherein said humanized antibody has an antigen binding affinityfor TAG-72 which is at least 10% that of CC49 and said humanizedantibody fragment has an amino acid sequence identical to that of aconstituent part of said humanized antibody.
 4. The humanized antibodyor humanized antibody fragment of claim 2 wherein said humanizedantibody has an antigen binding affinity for TAG-72 which is at least25% that of CC49.
 5. The humanized antibody or humanized antibodyfragment of claim 2 wherein said murine antibody is CC49, CC83, CC46,CC92, CC30, or CC11.
 6. The humanized antibody or humanized antibodyfragment of claim 5 wherein said murine antibody is CC49.
 7. Thehumanized antibody or humanized antibody fragment of claim 6 whereinsaid humanized antibody is expressed by ATCC CRL-12209 and saidhumanized antibody fragment has an amino acid sequence identical to thatof a constituent part of the antibody expressed by ATCC CRL-12209. 8.The humanized antibody or humanized antibody fragment of claim 1comprising a humanized variable heavy chain sequence of FIG. 1 or 3 or ahumanized variable light chain sequence of FIG. 2 or 4, or comprisingboth said humanized variable heavy chain sequence and said humanizedvariable light chain sequence.
 9. A nucleic acid sequence from which maybe expressed a humanized antibody or humanized antibody fragmentaccording to claim
 2. 10. A vector comprising a nucleic acid sequenceaccording to claim
 9. 11. The vector according to claim 10 wherein saidvector is a bare nucleic acid segment, a carrier-associated nucleic acidsegment, a nucleoprotein, a plasmid, a virus, a viroid, or atransposable element.
 12. A composition suitable for the treatment ofcancer comprising a therapeutically effective amount of a humanizedantibody or humanized antibody fragment according to claim
 1. 13. Thecomposition of claim 12 wherein said humanized antibody or humanizedantibody fragment is, directly or indirectly, associated with or linkedto an effector moiety having therapeutic activity, and the compositionis suitable for the treatment of cancer.
 14. The composition of claim 13wherein said effector moiety is a radionuclide, therapeutic enzyme,anti-cancer drug, cytokine, cytotoxin, or anti-proliferative agent. 15.A composition suitable for the in vivo or in vitro detection of cancercomprising a diagnostically effective amount of a humanized antibody orhumanized antibody fragment according to claim
 1. 16. The composition ofclaim 15 wherein said humanized antibody or humanized antibody fragmentis, directly or indirectly, associated with or linked to a detectablelabel, and the composition is suitable for detection of cancer.
 17. Thecomposition of claim 16 wherein the detectable label is a radionuclideor an enzyme.
 18. A method for in vivo treatment of a mammal having aTAG-72-expressing cancer comprising a step of administering to themammal a therapeutically effective amount of a composition according toclaim
 12. 19. A method for in vitro immunodetection of TAG-72-expressingcancer cells comprising a step of contacting the cancer cells with acomposition according to claim
 15. 20. The method of claim 19 whereinthe humanized antibodies or humanized antibody fragments of thecomposition are bound to a solid support.
 21. A method of in vivoimmunodetection of TAG-72-expressing cancer cells in a mammal comprisinga step of administering to the mammal a diagnostically effective amountof a composition according to claim
 15. 22. The method of claim 21wherein said immunodetection is in vivo tumor imaging.
 23. A method ofin vivo treatment of cancer comprising the steps of (i) intravenouslyadministering a radionuclide-labeled antibody, (ii) thereafter detectingtumor cells using a radionuclide activity probe, and (iii) thereafterremoving the detected tumor cells by surgical excision, wherein theantibody is a humanized antibody or humanized antibody fragmentaccording to claim
 1. 24. The method of claim 23, wherein theradionuclide is ¹²⁵I or 131I.